TWI380830B - Bendamustine pharmaceutical compositions - Google Patents

Description

Bendamustine pharmaceutical composition

The present invention relates to the field of pharmaceutical compositions for the treatment of various conditions, particularly tumorigenic and autoimmune diseases. In particular, the invention relates to pharmaceutical formulations comprising nitrogen mustard, particularly nitrogen mustard, bendamustine, such as bendamustine HCl.

Background of the invention

The present invention claims the benefit and priority of U.S. Application Serial No. 60/644,354, filed on Jan. 14, 2005, entitled "Bendamustine Pharmaceutical Composition", the full content (including schema and patent application scope) ) is incorporated herein by reference.

The following description includes information that can be used to understand the present invention. It is not an admission that such information is prior art to or in connection with the present invention, or any of the publications that are specifically or implicitly incorporated by reference.

Nitrogen mustard is highly reactive in aqueous solution, so it is difficult to formulate it into a pharmaceutical product, and it is often provided in a lyophilized form that needs to be reconstituted (usually in water) by a skilled medical staff before administration. . Once dissolved in an aqueous solution, the nitrogen mustard is degraded by hydrolysis, and therefore, the reconstituted product must be administered to the patient as soon as possible after its recovery.

Bendamustine (4-{5-[bis(2-chloroethyl)amino]-1-methyl-2-benzimidazolyl}butyric acid) is a benzimidazole ring An amorphous structure whose structure comprises an active nitrogen mustard (see Formula I, which lists bendamustine hydrochloride).

Bendamustine was originally synthesized in East Germany (GDR) in 1963 and sold under the trade name Cytostasan® in the country between 1971 and 1992. Since then, it has been sold in Germany under the trade name Cytostasan®. It has been widely used in Germany for the treatment of chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma and breast cancer.

Since bendamustine is degraded in aqueous solution (similar to other nitrogen mustards), it is provided as a lyophilized product. The bendamustine lyophilized formulation (Ribomustin®) used today contains bendamustine hydrochloride and mannitol in a sterile, lyophilized form in the form of a white powder for intravenous administration after reconstitution. The finished lyophilized product is unstable when exposed to light. Therefore, store the product in a brown or amber glass bottle. Existing bendamustine lyophilized formulations contain degradation products that may be produced during the manufacture of the drug, and/or during the lyophilization process of the final drug.

Currently, bendamustine is formulated as a lyophilized powder for injection of 100 mg of drug per 50 mL vial or 25 mg of drug per 20 mL vial. The vial is opened and restored as close as possible to the patient's time of administration. The product was reconstituted with sterile water for injection in 40 mL (for 100 mg form) or 10 mL (for 25 mg form). The reconstituted product was further diluted to 500 mL (q.s.) 0.9% sodium chloride solution for injection. The route of administration is via intravenous infusion for 30 to 60 minutes.

After recovering with 40 mL of sterile water for injection, the bendamustine vial can be stored at room temperature for 7 hours or at 6-8 ° C for 6 days. The 500 mL mixed solution must be reconstituted in the vial for 7 hours (assuming the mixture is stored at room temperature) and administered to the patient.

The recovery of the existing bendamustine lyophilized powder is quite difficult. Reports from the clinic indicate that recovery may take at least fifteen minutes and may take up to thirty minutes. In addition to being cumbersome and time consuming for the healthcare professional responsible for restoring the product, long-term exposure of bendamustine to water during reconstitution increases its loss of efficacy and the formation of impurities due to hydrolysis of the product by water. possibility.

Therefore, there is a need to develop bendamustine lyophilized formulations that are easier to recover and that have a better impurity profile than the existing bendamustine lyophilized (lyophilized powder) formulation.

German (GDR) Patent No. 34727 discloses a preparation of a 1-position substituted ω-[5-bis-(β-chloroethyl)-amino-benzimidazolyl-(2)]-alkanecarboxylic acid. method.

German (GDR) Patent No. 80967 discloses a γ-[1-methyl-5-bis-(β-chloroethyl)-amino-benzimidazolyl-(2)]-butyric acid hydrochloride Inject the preparation.

German (GDR) Patent No. 159,877 discloses a process for the preparation of 4-[1-methyl-5-bis-(β-chloroethyl)-amino-benzimidazolyl-2]-butyric acid.

German (GDR) Patent No. 159289 discloses an injectable solution of bendamustine.

Ribomustin® Bendamustine Product Monograph (updated 1/2002) http://www.ribosepharm.de/pdf/ribomustinbedamustin/productmonograph.pdf provides information about Ribomustin®, including product descriptions.

Ni et al. reported that nitrosourea SarCNU is stable in pure tert-butanol compared to pure acetic acid, dimethyl hydrazine, methanol, water or in a TBA/water mixture (Ni et al. (2001) Intl. J. Phamaceutics 226 :39-46).

Lyophilized cyclophosphamide is known in the art, for example, in U.S. Patent Nos. 5,418,233; 5,413,995; 5,268,368; 5,227,374; 5,130,305; 4,659,699; 4,537,883 and 5,066,647.

Lyophilized nitrogen mustard isocyclic phosphoniumamines are disclosed in International Patent Publication No. WO 2003/066027; U.S. Patent Nos. 6,613,927; 5,750,131; 5,972,912; 5,227,373 and 5,204,335.

Teagarden et al. disclose a lyophilized formulation of prostaglandin E-1 prepared by dissolving PGE-1 in a solution containing lactose and a third butanol (U.S. Patent No. 5,770,230).

The present invention relates to a stable pharmaceutical composition for nitrogen mustard, especially lyophilized bendamustine, and to the use thereof for the treatment of various conditions, particularly tumorigenic diseases and autoimmune diseases.

A particular aspect of the invention is a pharmaceutical composition of bendamustine containing no more than about 0.5% (by area of bendamustine) HP1 (as shown in Formula II), Formula II

Wherein the amount of HP1 is the amount of HP1 present at time point 0 upon release, or after reconstitution of the bendamustine lyophilized pharmaceutical composition of the present invention. A preferred embodiment is one containing no more than about 0.45% (by area percentage of bendamustine) HP1, preferably no more than about 0.40%, more preferably no more than about 0.35%, more preferably Not more than about 0.30%, even more preferably no more than about 0.25% of the bendamustine pharmaceutical composition.

Another embodiment of the invention is a benzene comprising at least more than about 0.1% to about 0.3% bendamustine dimer (as shown in Formula III) at the time of release or at time point after recovery. Damustine lyophilized preparation

Still another embodiment of the present invention is a method comprising not more than about 0.5% (preferably 0.15% to about 0.5%) of bendamustine ethyl ester at the time of release or at a time point after recovery. Bendamustine lyophilized formulation as shown in Formula IV)

Yet another embodiment of the present invention is a method for freeze-drying a bendamustine An agent wherein the concentration of bendamustine ethyl ester (as shown in Formula IV) is no more than about 0.2% (preferably 0.1%) greater than the benzoic acid present in the drug body used to make the lyophilized formulation Moustin ethyl ester concentration.

Another embodiment of the invention is a lyophilized formulation of bendamustine containing no more than about 0.5% (by area of bendamustine) HP1 when the drug is released. A preferred embodiment is one containing no more than about 0.45% (by area percentage of bendamustine) HP1, preferably no more than about 0.40%, more preferably no more than about 0.35%, more preferably Not more than about 0.30%, even more preferably no more than about 0.25% of the bendamestine lyophilized formulation. One aspect of this embodiment is a benzoxastatine lyophilized formulation containing no more than about 0.5% (by area percentage of bendamustine) HP1 when the drug is released, wherein the lyophilized formulation is packaged In small vials or other pharmaceutically acceptable containers.

In yet another aspect of the invention, with respect to the HP1 content, the bendamustine lyophilized formulation can remain stable for at least about 6 months (preferably 12 when stored at about 2 ° C to about 30 ° C) Months, preferably 24 months) to about 36 months or more. A preferred storage temperature is about 5 ° C and about room temperature.

Another embodiment of the invention is that it comprises no more than about 0.5% HP1, preferably no more than about 0.45%, preferably no more than about 0.40%, more preferably no more than about 0.35%, more preferably no more than about 0.30%, even more preferably no more than about 0.25% of the pharmaceutical dosage form of the bendamustine pharmaceutical composition, wherein HP1 is upon release, or is reconstituted The amount of HP1 present at the initial time point after the bendamestine formulation of bendamustine. In a preferred aspect of the invention, the dosage form can be from about 5 to 500 mg of bendamus. T, about 10 to 300 mg of bendamustine, about 25 mg of bendamustine, about 100 mg of bendamustine, and about 200 mg of bendamustine.

Another embodiment of the invention is a pharmaceutical dosage form comprising a benzoxastatine lyophilized formulation containing no more than about 0.5% HP1. A preferred dosage form may be from about 5 to 500 mg of bendamustine, from about 10 to 300 mg of bendamustine, about 25 mg of bendamustine, about 100 mg of bendamustine, and about 200 mg. Bendamustine.

In still another embodiment, the invention includes a method comprising comprising no more than about 0.5% (area percent by area of bendamustine), preferably no more than about 0.45%, preferably no more than About 0.40%, more preferably no more than about 0.35%, more preferably no more than about 0.30%, even more preferably no more than about 0.25% of bendamustine of HP1, and a trace amount of one or more organic solvents The bendamustine pharmaceutical composition, wherein the HP1 is the amount of HP1 present at time point 0 after release, or after reconstitution of the bendamustine lyophilized pharmaceutical composition as disclosed herein. In various aspects of this embodiment, the organic solvent is selected from the group consisting of one or more of third butanol, n-propanol, n-butanol, isopropanol, ethanol, methanol, acetone, ethyl acetate, dimethyl carbonate, Acetonitrile, dichloromethane, methyl ethyl ketone, methyl isobutyl ketone, 1-pentanol, methyl acetate, carbon tetrachloride, dimethyl hydrazine, hexafluoroacetone, chlorobutanol, dimethyl hydrazine , acetic acid and cyclohexane. Preferred organic solvents include one or more of ethanol, methanol, propanol, butanol, isopropanol and tert-butanol. A more preferred organic solvent is tert-butanol, also known as TBA, t-butanol, tert-butanol or tert-butanol.

The present invention encompasses a method of obtaining a license from a regulatory authority for obtaining bendamustine products, the improvement comprising the specification of release of bendamustine degradation products It is set to less than about 4.0%, preferably from about 2.0% to about 4.0% (by the area percentage of bendamustine), or otherwise to achieve the pharmaceutical composition of the present invention. One aspect of this particular aspect is a method for obtaining regulatory approval for a product of bendamustine comprising setting the release specification for HP1 to less than or equal to 1.5% (in areas of bendamustine) percentage). The bendamustine product herein contains no more than about 0.5% (by area percentage of bendamustine) HP1 when released.

Another embodiment is a method for obtaining regulatory approval for a bendamustine product, the improvement comprising setting a shelf life specification of bendamustine degradation product to less than about 7.0%, preferably From about 5.0% to about 7.0% (by area percentage of bendamustine), wherein the product is stored at about 2 ° C to about 30 ° C. A preferred storage temperature is about 5 ° C and about room temperature. The bendamustine product herein contains no more than about 0.5% (by area percentage of bendamustine) HP1 when released.

Another embodiment of the invention is a method for making a bendamestine lyophilized formulation comprising controlling the concentration of bendamustine degradant in the final product to render the bendamustine degradant The concentration is less than about 4.0%, preferably from about 2.0% to about 4.0% (by area percentage of bendamustine) upon release, or otherwise the pharmaceutical composition of the invention can be obtained. The bendamustine product herein contains no more than about 0.5% (by area percentage of bendamustine) HP1 when released.

The present invention discloses a method for producing a bendamustine lyophilized formulation comprising controlling the concentration of bendamustine degradant in the final product such that upon release, the concentration of HP1 is less than about 0.5% Bendamustine Percentage of area), and at the expiration of the expiration date of the product, the concentration of bendamustine degradant is less than about 7.0%, preferably no more than about 5.0% to about 7.0%; wherein the product is stored at about 2 °C to about 30 °C.

Another embodiment of the present invention is a bendamustine pre-lyophilized solution or dispersion comprising one or more organic solvents, wherein the solution or dispersion comprises at least one organic solvent having a stable concentration, which can be reduced The degree of degradation of bendamustine is such that the total amount of HP1 produced during lyophilization from 0 to 24 hours is no more than about 0.5% (by area percentage of bendamustine), preferably 0.45%, preferably The ground is 0.40%, more preferably 0.35%, more preferably 0.30%, even more preferably 0.25%. One aspect of this particular aspect is a lyophilized powder prepared from the pre-lyophilized solution or dispersion.

Still another embodiment of the present invention is a bendamustine pre-lyophilized solution or dispersion comprising one or more organic solvents, wherein the solution or dispersion comprises at least one organic solvent having a stable concentration, The degree of degradation of bendamustine can be reduced such that the total amount of bendamustine ethyl ester produced during lyophilization from 0 to 24 hours is no more than about 0.5% (by area percentage of bendamustine). One aspect of this particular aspect is a lyophilized powder prepared from the pre-lyophilized solution or dispersion.

Still another embodiment of the present invention is a bendamustine pre-lyophilized solution or dispersion comprising one or more organic solvents, wherein the solution or dispersion comprises at least one organic solvent having a stable concentration, which is Decreasing the degree of degradation of bendamustine such that the total amount of bendamustine ethyl ester (as shown in Formula IV) produced during lyophilization from 0 to 24 hours is no more than 0.2%, preferably 0.1%, Greater than the amount of bendam present in the drug body used to make the lyophilized formulation The concentration of ethyl ester. A preferred organic solvent is tert-butanol.

The invention also discloses a process for the preparation of a bendostatin lyophilized formulation comprising dissolving bendamustine in an alcohol having a stability concentration of between about 5% and about 100% (v/v alcohol) Forming a pre-lyophilized solution in a solvent-like solvent; and subjecting the pre-lyophilized solution to freeze-drying; wherein the bendamustine lyophilized preparation prepared from such a method contains no more than about 0.5% (in terms of benzene) Percentage of area of Mostin) HP1 as shown in Formula II, wherein the HP1 is the amount of HP1 present at the time point 0 after release, or after reconstitution of the bendamustine lyophilized pharmaceutical composition. Other alcohol concentrations include from about 5% to about 99.9%, from about 5% to about 70%, from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 20% to about 35%. Preferred concentrations of alcohols range from about 20% to about 30%. Preferred alcohols include one or more of methanol, ethanol, propanol, isopropanol, butanol and tert-butanol. A more preferred alcohol is tert-butanol. The preferred concentration of the third butanol is from about 20% to about 30%, preferably about 30%. One aspect of this particular aspect is the addition of excipients prior to lyophilization. A preferred excipient is mannitol. The preferred pre-lyophilized concentration of bendamustine is between about 2 mg/mL and about 50 mg/mL.

In a preferred method for preparing a bendamestine lyophilized formulation, the procedure of lyophilizing the pre-lyophilized solution comprises i) freezing the pre-lyophilized solution to less than about -40 ° C. a temperature of (preferably -50 ° C) to form a frozen solution; ii) maintaining the frozen solution at or below about -40 ° C (preferably -50 ° C) for at least 2 hours; iii) causing the knot The frozen solution is ramped to a first drying temperature between about -40 ° C to about -10 ° C to form a dry solution; iv) maintained for about 10 to about 70 hours; v) the drying solution is gradually increased to Between about 25 And a second drying temperature between ° C and about 40 ° C; and vi) maintaining from about 5 to about 40 hours to form a bend free formulation of bendamustine. In a preferred method, the step of lyophilizing the pre-lyophilized solution comprises i) freezing the pre-lyophilized solution to about -50 ° C to form a frozen solution; ii) maintaining the frozen solution At least about 2 hours to about 4 hours at about -50 ° C; iii) increasing the frozen solution to a first drying temperature of between about -20 ° C and about -12 ° C to form a dry solution; iv) maintaining 10 to about 48 hours at a first drying temperature; v) increasing the dried solution to a second drying temperature of between about 25 ° C and about 40 ° C; and vi) maintaining at a second drying temperature for at least 5 hours at most It takes about 20 hours. A more preferred alcohol is tert-butanol. The preferred concentration of the third butanol is from about 20% to about 30%, preferably about 30%. One aspect of this particular aspect is the addition of excipients prior to lyophilization. A preferred excipient is mannitol. The preferred pre-lyophilized concentration of bendamustine is between about 2 mg/mL and about 50 mg/mL.

Another embodiment of the invention is a lyophilized powder or formulation obtained by the process of preparing a bendamestine lyophilized formulation disclosed herein.

The present invention also encompasses a bendamustine formulation for lyophilization comprising an excipient and an organic solvent at a given concentration. Preferred formulations include bendamustine at a concentration of about 15 mg/mL, mannitol at a concentration of about 25.5 mg/mL, and a concentration of about 30% (v/v) tert-butanol and water. This particular aspect of the invention includes lyophilized formulations prepared from such bendamustine formulations.

The invention includes a method of treating a condition in a patient comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention, and wherein the condition is a therapeutic to the pharmaceutical composition. Certain conditions that can be treated by the compositions of the present invention Includes, chronic lymphocytic leukemia (CLL), Hodgkin's disease, non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), breast cancer, small cell lung cancer, hyperproliferative disorders, and Immune disease. Preferred conditions include NHL, CLL, breast cancer and MM. Preferred autoimmune diseases include rheumatoid arthritis, multiple sclerosis or lupus.

The present invention encompasses the use of a pharmaceutical composition or a pharmaceutical preparation of the present invention for the manufacture of a medicament for treating a condition in a patient (as defined above) comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention, and the condition is It is possible to treat the pharmaceutical composition.

The invention also includes a method of treating a pharmaceutical composition of the invention in combination with one or more anti-neoplastic agents, the anti-neoplastic agent being administered prior to, concurrently with, or subsequent to administration of the pharmaceutical composition of the invention. A preferred anti-tumor agent is an antibody specific for CD20.

Another embodiment of the invention is a freeze-drying cycle for making the freeze-dried bendamustine formulation of the invention. Preferably, the freeze-drying cycle comprises a) freezing to about -50 ° C over a period of about 8 hours; b) maintaining at -50 ° C for about 4 hours; c) gradually increasing to -25 ° C over a period of about 3 hours; d) maintained at -10 ° C for about 3 hours; e) gradually increased to about 25 ° C to about 40 ° C or higher over a period of about 3 hours; f) maintained between about 25 ° C to about 40 ° C About 25 hours; g) cooling to about 20 ° C over a period of 1 hour; h) discharging at a pressure of 13.5 psi at about 20 ° C in a pharmaceutically acceptable container, which is sealed; The pressure throughout the first drying is about 150 microns and is about 50 microns throughout the second drying. One aspect of this cycle consists of gradually increasing the temperature to about 30-35 ° C for 3 hours, then gradually increasing Step (e) of heating to 40 ° C for 5 hours. Another aspect of this particular aspect is a lyophilized powder prepared from such a freeze-drying cycle. A preferred freeze-drying cycle comprises i) feeding at a shelf temperature of about 5 ° C; ii) freezing to about -50 ° C over a period of about 8 hours; iii) maintaining at -50 ° C for about 4 hours; Iv) gradually increase to -20 °C over a period of about 3 hours; v) maintain at -20 ° C for 6 hours, gradually increase to -15 ° C over a period of about 1 hour; vi) maintain at -15 ° C About 20 hours; vii) gradually warming to -15 ° C over a period of about 1 hour; viii) maintained at -15 ° C for about 20 hours; ix) gradually warming to -12 ° C over a period of about 0.5 hours ; x) maintained at -12 ° C for about 15.5 hours; xi) gradually increased to about 25 ° C to about 40 ° C or higher over a period of about 15 hours; xii) maintained between about 25 ° C to about 40 ° C Up to about 10 hours; xiii) gradually warming to 40 ° C over a period of about 1 hour; and xiv) maintaining at 40 ° C for about 5 hours; at about 5 ° C, at 13.5 psi, discharging at the drug In an acceptable container, it is sealed; wherein the pressure throughout the first drying is about 150 microns and the second drying is about 50 microns. In a preferred embodiment, step (xi) is gradually ramped to about 30-35 ° C over a period of about 15 hours.

The invention also encompasses a pharmaceutical dosage form of bendamustine containing no more than about 0.5% HP1 (area percentage of bendamustine), wherein the dosage form comprises a vial or other pharmaceutically acceptable container Wherein HP1 is the amount of HP1 present prior to recovery, or at an initial point in time after the dosage form is restored. Preferred bendamustine concentrations include from about 10 to about 500 mg per container, about 100 mg per container, from about 5 mg to about 2 g per container, and about 170 mg per serving. container.

The invention also includes a pre-lyophilized pharmaceutical composition of bendamustine. Preferred pre-lyophilized compositions include bendamustine HCl of about 15 mg/mL, mannitol of about 25.5 mg/mL, and about 30% (v/v) of third butanol and water.

These and other aspects of the invention are described below, or may be apparent to those skilled in the art based on the following disclosure.

As used herein, the term "mixed" means that the preparation of a drug (eg, bendamustine) is in a form suitable for administration to a mammalian patient, preferably a human. Thus, a "formulation" can include the addition of a pharmaceutically acceptable excipient, diluent or carrier.

As used herein, the term "lyophilized powder" or "lyophilized formulation" means any solid material obtained by freeze drying, that is, by freezing-drying an aqueous solution. The aqueous solution may contain a non-aqueous solvent, that is, it is a solution consisting of aqueous and one or more non-aqueous solvents. Preferably, the lyophilized preparation is one in which the solid substance is obtained by freeze-drying a solution composed of an aqueous one and a plurality of non-aqueous solvents, and more preferably the non-aqueous solvent is an alcohol.

"Deeping pharmaceutical composition" means any pharmaceutical composition that has sufficient stability to have a use as a pharmaceutical product. Preferably, the tranquil pharmaceutical composition has a stability sufficient to be stored at a convenient temperature (preferably between -20 ° C and 40 ° C, more preferably from about 2 ° C to about 30 ° C) for a reasonable period of time, for example The shelf life of the product can be as short as one month, but representatively six months or longer, more preferably one year or longer, or even better two Fourteen months or longer, and even more preferably thirty-six months or longer. The shelf life or expiration date can be a time point at which the active ingredient degrades to less than 90% purity. For the purposes of the present invention, a tranquil pharmaceutical composition comprises a pharmaceutical composition having a specific impurity range as described herein. Preferably, the stable pharmaceutical composition is one of which has the lowest active ingredient degradation, that is, it remains at least about 85% (preferably at least about 90%) over a period of 2-3 years at 2-30 °C. And more preferably at least about 95%) of the undegraded active ingredient.

By "diazea lyophilized formulation" is meant any utility that has sufficient stability, for example, having characteristics similar to those defined herein for a pharmaceutical composition to have a lyophilized formulation that can be used as a pharmaceutical product.

"Degraded" means that the active ingredient has undergone a chemical structural change.

The term "therapeutically effective amount" as used herein, means that the compound to be administered can reduce one or more of the symptoms of the condition to some extent. Regarding the treatment of tumors, a therapeutically effective amount means having (1) reducing tumor size, (2) inhibiting (i.e., slowing down to a certain extent, preferably stopping) tumor metastasis, and (3) inhibiting tumor growth to a certain extent. (i.e., slowing down to some extent, preferably stopping), and/or (4) reducing the effectiveness of one or more of the symptoms associated with the cancer to a certain degree (or preferably eliminated). A therapeutically effective amount may also mean preventing the disease (preventive treatment) from occurring in an animal that may be susceptible to the disease but has not experienced or presented the symptoms of the disease. Further, a therapeutically effective amount can be an amount that increases the survival expectation of a patient suffering from a terminal disease. For the treatment of non-Hodgkin's lymphoma with bendamustine, a representative therapeutically effective dose may be about 60-120 mg/m ² for two consecutive days in unit dosage form. This cycle can be repeated every three to four weeks. For the treatment of chronic lymphocytic leukemia (CLL), bendamustine at a dose of about 80-100 mg/m ² can be administered on days 1 and 2. This cycle can be repeated after about 4 weeks. For the treatment of Hodgkin's disease, bendamustine can be administered at a dose of 25 mg/m ² of daunorubicin on days 1 and 15, and a dose of 10 mg on days 1 and 15. m ² of bleomycin, on day 1, and 15 administered at a dose of 1.4 mg / m ² of vincristine, and administered on days 1-5 in a dose of 50 mg / m ² of benzene bendamustine This is done, and the "DBVBe treatment" of the cycle is repeated about every 4 weeks. For breast cancer, bendamustine (120 mg/m ² ) can be administered on days 1 and 8, in combination with a dose of 40 mg/m ² of methotrexate on days 1 and 8, and The 5-fluorouracil was administered at a dose of 600 mg/m ² on days 1 and 8, and the cycle was repeated approximately every 4 weeks. As a second-line breast cancer therapy, bendamustine at a dose of 100-150 mg/m ² can be administered on days 1 and 2.

As used herein, "tumorigenicity" means an abnormally growing tumor that occurs as a result of cell proliferation that does not generally limit growth. As used herein, "anti-tumor agent" refers to any compound, composition, mixture, co-mix or blend that inhibits, eliminates, delays or reverses the tumorigenic phenotype of a cell.

As used herein, "hyperproliferation" refers to the excessive production of cells by response to specific growth factors. A "hyperproliferative disease" is a disease in which cells are overproduced by reaction to a particular growth factor. Examples of such "hyperproliferative diseases" include diabetic retinopathy, cowhide, endometriosis, cancer, macular degeneration, and benign growth diseases, such as the forefront Glandular hypertrophy.

As used herein, the term "small vial" means any closable container, whether hard or soft.

"Control" as used herein means the application of appropriate process controls to assist in the event of an event to be controlled. For example, in the case given, "control" may mean testing individual or many samples regularly or randomly; setting the concentration of degradation products to release specifications; selecting treatment conditions, such as pre-lyophilized solutions or Alcohols and/or other organic solvents are used in the dispersion to ensure that the concentration of the degradation of the active ingredient is not unacceptably high; Controlling degradants by setting a release specification for the amount of degradation products can help the pharmaceutical products to be issued by regulatory authorities (such as the US Food and Drug Administration and similar agencies in other countries or regions ("Bureau"). Control license.

The term "pharmaceutically acceptable" as used herein means that it is pharmaceutically acceptable, for example, a component (including a container) in a pharmaceutical composition that does not cause unacceptable loss of pharmacological activity or unacceptable side effects. . Examples of pharmaceutically acceptable components can be found in the United States Pharmacopoeia (USP), the National Pharmacopoeia (NF), in the United States Drugs Agreement, in Rockville, Md. in 1990, and the FDA Active Ingredient Guide 1990, 1996 by US Food and Published by the Drug Administration (all references are incorporated herein by reference, including any drawings). Other grades of solutions or components that meet requirements beyond the USP/NF limits and/or specifications may also be used.

The term "pharmaceutical composition" as used herein shall mean a composition made under conditions which render it suitable for administration to humans, for example, it is prepared under GMP conditions and contains pharmaceutically acceptable excipients. For example (but not limited to) Stabilizers, fillers, buffers, carriers, diluents, carriers, cosolvents and binders. Pharmaceutical compositions for use herein include, but are not limited to, pre-lyophilized solutions or dispersions, as well as liquid forms for injection or perfusion immediately after reconstitution of the lyophilized preparation.

"Pharmaceutical dosage form" as used herein is meant to be present in a container as disclosed herein and in an amount suitable for reconstitution and one or more doses (representatively about 1-2, 1-3, 1-4, 1) a pharmaceutical composition for administration of -5, 1-6, 1-10 or about 1-20 doses. Preferably, "medical dosage form" as used herein is meant to be present in a container as disclosed herein, and in an amount suitable for reconstitution and one or more doses (representatively about 1-2, 1-3, 1 -4, 1-5, 1-6, 1-10 or about 1-20 doses of the delivered pharmaceutical composition. The pharmaceutical dosage form can comprise a vial or syringe or other suitable pharmaceutically acceptable container. Pharmaceutical dosage forms suitable for injectable or perfusion may include sterile aqueous solutions or dispersions or sterile powders containing active ingredients suitable for the preparation of sterile injectable or perfusible solutions or dispersions. In summary, the final dosage form is sterile, fluid, and is stable under the conditions of manufacture and storage. The liquid carrier or carrier can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, polyols such as glycerol, propylene glycol or liquid polyethylene glycol, vegetable oils, non-toxic glycerides, and suitable mixtures thereof. Microbial growth can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

As used herein, the term "excipient" means a substance used to formulate an active pharmaceutical ingredient (API) into a pharmaceutical formulation; in a preferred embodiment, the excipient does not reduce or interfere with the primary therapeutic efficacy of the API. . Preferably, the excipient It is inert to treatment. The term "excipient" encompasses carriers, diluents, carriers, solubilizers, stabilizers, fillers, and binders. Excipients can also be those which are present in the pharmaceutical formulation due to indirect or unexpected results of the manufacturing process. Preferably, the excipient is approved for use or is considered safe for humans and animals, i.e., GRAS material (generally considered safe). GRAS substances are listed by the US Food and Drug Administration as 21 CFR § 182 and 21 CFR § 184 of the Federal Regulations (CFR), which are incorporated herein by reference. Preferred excipients include, but are not limited to, hexitols, including mannitol and the like.

As used herein, "to make a stable concentration of an organic solvent" or "a stable concentration of an alcohol" means that the amount of organic solvent or alcohol can reduce the degree of degradation of bendamustine to achieve the final drug product as defined. Degradant concentration. For example, with respect to the degradation product HP1, a concentration of the organic solvent in an amount such that the HP1 concentration (in terms of the area percentage of bendamustine) is less than about 0.5%, preferably less than 0.45%, preferably less than 0.40%, more preferably less than 0.35%, more preferably less than 0.30%, and even more preferably less than 0.25%. With regard to the overall or overall concentration of the final drug product, the amount of organic solvent in a given concentration may result in a total degradant concentration (at the time of release of the drug product) of less than about 7% (by area percentage of bendamustine), Preferably less than about 6%, preferably less than about 5%, and even more preferably less than about 4%. "Percent area by bendamustine" means the amount of a specific degradant (e.g., HP1) relative to the amount of bendamustine when measured by HPLC.

The term "organic solvent" means an organic substance, usually a liquid, which is capable of dissolving other substances.

As used herein, "microorganic solvent" means that it is equivalent to or lower than the recommended concentration for use in a pharmaceutical product, such as by the ICH indicator (International Harmony Conference, Impurity - Residual Solvent Indicators. Q3C. Federal Records. 1997; 62 (247): 67377) Suggested. The bottom limit is the lowest amount that can be detected.

The term "release" or "at release" means that the pharmaceutical product has met the release specifications and is useful for the medical purpose it is intended to achieve.

A. General

The present invention provides a stable, pharmaceutically acceptable composition prepared from bendamustine. In particular, the present invention provides freeze-dried formulations for bendamustine HCl. The lyophilized powder obtained from such formulations is easier to recover than the currently available bendamustine lyophilized powder. Moreover, in terms of specific impurities (especially HP1, bendamustine dimer and bendamustine ethyl ester), the lyophilized product of the present invention is reconstituted, reconstituted or mixed during lyophilization or during reconstitution and mixing. After that, it has a better impurity distribution than Ribomustin®.

The invention further provides a bendamustine formulation for use in the treatment of a neoplastic disease. The formulations described herein can be administered alone or in combination with at least one additional anti-neoplastic agent and/or radiation therapy.

One aspect of the invention is a condition and method for enhancing the stability of bendamustine during storage and during recovery prior to and during the freeze-drying process.

Antineoplastic agents useful in combination with the formulations of the present invention include those listed in Merck Index, 11th Edition, pp 16-17, Merck & Co., Inc. (1989) and Chemotherapy Source Books (1997). These two books are well known to those skilled in the art and are readily available.

There are a number of anti-neoplastic agents available for commercial use, clinical evaluation, and preclinical development that can be selected for cancer treatment by combination chemotherapy. Such anti-tumor agents are divided into several major classes, namely, antibiotic-type agents, covalent DNA-binding drugs, antimetabolite agents, hormone agents (including glucocorticoids such as prednisone and dexamethasone), immunization Agents, interferon-type agents, differentiation agents such as retinoids, pro-apoptotic agents, and miscellaneous agents include compounds such as antisense, small interfering RNA, and the like. Alternatively, other anticancer agents can be used, such as metalloproteinase (MMP) inhibitors, SOD mimetics or avar vet beta ₃ inhibitors.

A family of anti-neoplastic agents useful in combination with the compounds of the invention consists of an antimetabolite-type agent. Suitable anti-metabolite anti-neoplastic agents are selected from alanosine, AG2037 (Pfizer), 5-fibrinogen (5-FU-fibrinogen), acanthifolic acid, aminothiazepine Oxazole, brequinar sodium, carmofur, Ciba-Geigy CGP-30694, cyclopentylcytosine, cytarabine stearate, cytarabine conjugate , Lilly DATHF, Merrel Dow DDFC, dezaguanine, dedeoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, dexifluuridine ), Wellcome EHNA, Merck & Co. EX-105, fazarabine, floxuridine, fludarabine, 5-fluorouracil, N-(2'-tetrahydrofuranyl -5-fluorouracil, first pharmaceutical FO-152 (Daiichi Seiyaku FO-152), isopropylpyrroline Lilly LY-188011, Lilly LY-264618, metobazoprim, methotrexate, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC- 39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788 (Takeda TAC) -788), thioguanine, tiazofurin, Erbamont TIF, trimetrexate, tyrosine kinase inhibitor, tyrosine protein kinase inhibitor, Dapeng UFT (Taiho UFT) and You Yousheng (unicytin).

The second family of anti-neoplastic agents useful in combination with the compounds of the invention consists of a covalent DNA-binding agent. Suitable alkylating-type anti-tumor agents may be selected from the group consisting of: salt wildwood 254-S (Shionogi 254-S), aldehyde-phosphonium analog, altretamine, anazirone, Boehringer Mannheim BBR -2207, bestrabucil, budotitane, and long-length CA-102 (Wakunaga CA-102), carboplatin, carmustine, Chinoin-139, Chinoin-153, phenylbutyrate Chlorambucil, cisplatin, cyclophosphamide, American cyanamide CL-286558, Sanofi CY-233, cyplatate, Degussa D-19-384, DACHP(Myr) ₂ , diphenyl snail Diphenylspiromustine, diplatinum cytostatic, Erba distamycin derivative, Chugai DWA-2114R, ITI E09, emmustine, Erbamont FCE-24517, female Estramustine sodium phosphate, fotemustine, Unimed G-6-M, Chinoin GYKI-17230, hepsul-fam, ifosfamide, iproplatin, lo Lomustine, mafosfamide, melphalan, mitolactol, NK-1 21, NCI NSC-264395, NCI NSC-342215, oxaliplatin, Upjohn PCNU, prednimustine, Proter PTT-119, rarimustine, and semesterine Semustine), SmithKline SK&F-101772, Yakult Honsha SN-22, spiromustine, Tanabe Pharmaceutical TA-077, tauromustine, temozolomide, teroxirone, Tetraplatin and trimelamol.

Another family of anti-neoplastic agents that can be used in combination with the compounds of the invention consists of an antibiotic-like anti-neoplastic agent. Suitable antibiotic-type antitumor agents can be selected from Dapeng 4181-A, aclarubicin, actinomycin D, actinoplanone, alanosine, Erbamont ADR-456, gas Aleoplysinin derivative, Ajinomoto AN-201-II, Ajinomoto AN-3, Anisomycins, Anthracycline, A mycin-A, bicucapiern, Bristol-Myers BL-6859, Bristol-Myers BMY-25067, Bristol-Myers BMY-25551, Bristol-Myers BMY-26605, Bristol-Myers BMY-27557, Bristol-Myers BMY-28438, sulfuric acid Bleomycin sulfate, bryostatin-1, Dapeng C-1027, calichemycin, chromoximycin, dactinomycin, daunorubicin, Kyowa Hakko DC-102, Kyowa Hakko DC-79 , Kyowa Hakko DC88A, Kyowa Hakko DC89-A1, Kyowa Hakko DC92-B, ditrisarubicin B, salt wildwood DOB-41, doxorubicin, doxorubicin-fibrinogen , elsamicin-A, epirubicin, erbstatin, esorubicin, esperamicin-A1, esperamicin-A1b, Erbamont FCE-21954, Fujisawa FK-973, Fostricin, vinegar FK-900482, glidobactin, gregatin-A, grincamycin, herbimycin, idarubicin, illudins, kazusamycin, kesarirhodins, Kyowa Hakko KM-5539, Kirin Brewery KRN-8602, Kyowa Hakko KT-5432, Kyowa H Akko KT-5594, Kyowa Hakko KT-6149, American cyanamide LL-D49194, Meiji results ME 2303, menogaril, mitomycin, mitoxantrone, SmithKline M- TAG, neoenactin, Nippon chemical NK-313, Nippon chemical NKT-01, SRI international NSC-357704, oxalysine, oxaunomycin, peplomycin, pilatin, pirarubicin ), porothramycin, pyriddamycin A, Tobishi RA-I, rapamycin, rhizoxin, rodorubicin, sibanomicin, siwenmycin, Sumitomo SM-5887, Snow Seal SN-706, Snow Seal SN-07, sorangicin-A, sparsomycin, SS Pharmaceutical SS-21020, SS Pharmaceutical SS-7313B, SS Pharmaceutical SS-9816B, Steffimycin B, Dapeng 4181-2, talisomycin, TAN-868A, terpentecin, thrazine, trirozarin A, Upjohn U-73975, Kyowa Hakko UCN-10028A, Fujisawa WF-3405, Jifu Y-25024 and Zorobidin Zorubicin).

A fourth group of anti-neoplastic agents useful in combination with a compound of the invention includes a miscellaneous anti-neoplastic agent selected from the group consisting of alpha-carotene, alpha-difluoromethyl-arginine, aruqu Acitretin, arsenic trioxide, Avastin® (bevacizumab), Biotec AD-5, Kyorin AHC-52, alstonine, amonafide, amphethinile, amsacrine, Angiostat, ankinomycin, anti-tumor ketone A10, anti-tumor ketone A2, anti-tumor ketone A3, anti-tumor ketone A5, anti-tumor ketone AS2-1, Henkel APD, aphidicolin glycinate, aspartate, Avarol, baccharin ,batracylin, benfluron, benzotript, Ipsen-Beaufour BIM-23015, bisantrene, Bristol-Myers BMY-40481, Vestar boron-10, bromothioguanamine (bromofosfamide), Wellcome BW-502, Wellcome BW-773, caracemide, carmethizole hydrochloride, ajinophanes CDAF, chlorosulfaquine Chlorsulfaquinoxalone, Chemes CHX-2053, Chemes CHX-100, Warner-Lambert CI-921, Warner-Lambert CI-937, Warner-Lambert CI-941, Warner-Lambert CI-958, clanfenur , clavididone, ICN compound 1259, ICN compound 4711, Contracan, Yakult Honsha CPT-11, cristatol, curaderm, cytochalasin B, cytarabine , cytocytin, Merz D-609, DABIS maleate, Dacaba (dacarbazine), datelliptinium, didemnin-B, dihaematoporphyrin ether, dihydrolenperone, dinaline, distamycin, Toyo Pharmar DM-341, Toyo Pharmar DM-75, first pharmaceutical DN-9693, elliprabin, elliptinium acetate, epothiones, Tsumura EMTTC, erbitux, ergotamine, erlotnib, etoposide ( Etoposide), etretinate, fenretinide, vinegar FR-57704, strontium nitrate, genkwadaphnin, Gleevec® (imatnib), Chugai GLA-43, Glaxo GR-63178, gefitinib, Grifola Grifolan NMF-5N, hexadecane choline phosphate, Green Cross HO-221, homoharringtonine, hydroxyurea, BTG ICRF-187, indanocine, ilmofosine ), isoglutamic acid, isotretinoin, 冢JI-36, Ramot K-477, 冢K-76COONa, Kureha chemistry K-AM, MECT Corp KI-8110, American cyanamide L-623, white blood cells Regulators, lonidamine, Lundbeck LU-23-112, Lilly LY-186641, NCI (US) MAP, marycin, mefloquine, Merrel Dow MDL-27048, Medco MEDR-340, merbarone, merocyanine derivative, methylanilinoacridine, molecular genetics MGI-136, minactivin, Mitonafide, mitoquidone, mopidamol, motretinide, Zenyaku Kogyo MST-16, N-(astragalo) amino acid, Nisshin Flour Milling N-021, N-deuterated dehydroxyalanine, nafazatrom, Taisho NCU-190, nocodazole derivatives, Normosang, NCI NSC-145813, NCI NSC-361456, NCI NSC-604782, NCI NSC-95580, somatostatin octreotide, Ono ONO-112, oquizanocine, Akzo Org-10172, paclitaxel, pancreatic inhibin, pazelliptine, Warner-Lambert PD- 111707, Warner-Lambert PD-115934, Warner-Lambert PD-131141, Pierre Fabre PE-1001, ICRT peptide D, piroxantrone, polyhaematoporphyrin, polypreic acid ), Efamol porphyrin, probimane, procarbazine, progula (proglumide), Invitron protease nexin I, Tobishi RA-700, razoxane, Sapporo Breweries RBS, restricin-P, retelliptine, retinoic acid, Rhone-Poulenc RP-49532, Rhone- Poulenc RP-56976, Rituxan® (with other anti-CD20 antibodies such as Bexxar®, Zevalin®), SmithKline SK&F-104864, statins (Lipitor®, etc.), Sakamoto SM-108, Kuraray SMANCS, SeaPharm SP-10094, spatol , spirocyclopropane derivatives, spirogermanium, Unimed, SS pharmaceutical SS-554, strypoldine, Stypoldione, Suntory SUN 0237, Suntory SUN 2071, archaeal mustard, thalidomide, sand Lidoamine analogue, Toyama T-506, Toyama T-680, paclitaxel, Teijin TEI-0303, teniposide, thaliblastine, Eastman Kodak TJB-29, tocotrienol, totemistine (Topostin), Teijin TT-82, Kyowa Hakko UCN-01, Kyowa Hakko UCN-1028, ukrain, Eastman Kodak USB-006, vinblastine sulfate, vincristine, vinblastine, vinorelbine, vinorelbine, Changchun Quinol, vinorelbine, withanoli Des and YM-534 (Zometa®) in the mountains.

Examples of radiopharmaceuticals that can be used in combination with the chemotherapy of the present invention are AD-5, adchnon, amifostine analog, detox, dimesna, 1-102, MM-159, N-deuterated dehydroxyalanine, TGF-Genetech , tiprotimod, amifostine, WR-151327, FUT-187, ketoprofen transdermal, nabumetone, superoxide dismutase (Chiron and Enzon).

Methods for preparing the above antitumor agents can be found in the literature. For example, a method of preparing doxorubicin is described in U.S. Patent Nos. 3,590,028 and 4,012,448. Methods for preparing metalloproteinase inhibitors are described in EP 780386. For the preparation of the alpha v beta ₃ inhibitors are by method described in WO 97/08174.

Preferred anti-neoplastic agents include, but are not limited to, daunorubicin, bleomycin, vincristine, doxorubicin, dacarbazine, Prednisolone, mitoxantrone, prednisone, methotrexate, 5-fluorouracil, dexamethasone, thalidomide, Thalidomide derivatives, 2ME2, Neovastat, R 11 5777, arsenic trioxide, bortezomib, tamoxifen, G3139 (antisense) and SU5416, mitomycin, anti-CD20 antibodies such as Rituxan® and R- One or more of etodolac.

Preferred pharmaceutical procedures that can be used in conjunction with, or in place of, one or more of the components of the present invention include, but are not limited to, ABDV (doxorubicin, bleomycin, vincristine, dacarbazone), DBV (daunorubicin, bleomycin, vincristine), CVPP (cyclophosphamide, vinblastine, procarbazine, prednisolone), COP (cyclophosphamide, vincristine, prednisolone) Songlong), CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) and CMF (cyclophosphamide, methotrexate, 5-fluorouracil). Other treatments are listed in Table A below.

As described herein, the lyophilized formulation of bendamustine is achieved after removal of the organic solvent in water. The most representative example of a solvent used to prepare such formulations is third butanol (TBA). Other organic solvents that may be used include ethanol, n-propanol, n-butanol, isopropanol, ethyl acetate, dimethyl carbonate, acetonitrile, dichloromethane, methyl ethyl ketone, methyl isobutyl ketone. , acetone, 1-pentanol, methyl acetate, methanol, carbon tetrachloride, dimethyl hydrazine, hexafluoroacetone, chlorobutanol, dimethyl hydrazine, acetic acid, cyclohexane. These aforementioned solvents may be used singly or in combination. The solvent that can be used must form a stable solution with bendamustine and must not degrade or deactivate the API. The solubility of bendamustine in the solvent of choice must be high enough to form a commercially available concentration of the drug dissolved in the solvent. In addition, the solvent should be able to easily get from the drug Removal of the aqueous dispersion or solution of the product, for example by freeze drying or vacuum drying. Preferably, it is used at a concentration of from about 2 to 80 mg/mL, preferably from about 5 to 40 mg/mL, more preferably from 5 to 20 mg/mL, and even more preferably from 12 to 17 mg/mL. A solution of damostatin.

A pharmaceutically acceptable lyophilized excipient can be dissolved in the aqueous phase. Examples of excipients that can be used in the present invention include, but are not limited to, sodium or potassium phosphate, citric acid, tartaric acid, gelatin, glycine, and hydrocarbons such as lactose, sucrose, maltose, glycerin, dextrose, paste. Fine, trehalose and hydroxyethyl starch. Mannitol is a preferred excipient. If desired, other excipients may be used, including antioxidants such as (not limited to) ascorbic acid, acetylcysteine, cysteine, sodium hydrogen sulfate, butyl-hydroxyanisole, butyl-hydroxytoluene or Ava-tocopheryl acetate, or a chelating agent.

Representative formulations that can be used in accordance with the present invention, and freeze-drying cycles are listed below. Freeze drying can be accomplished using standard equipment such as for freeze drying or vacuum drying. The cycle may vary depending on the device and equipment used for filling/finishing.

According to a representative embodiment of the invention, the pre-lyophilized aqueous solution or dispersion is first formulated in a pharmaceutically acceptable mixing container. The solution is sterile filtered into a sterile container, filled into vials of appropriate size, partially plugged and placed in a freeze dryer. The solution is lyophilized using the freeze drying technique of the present invention until the moisture content is in the range of from about 0.1 to about 8.0%. The resulting lyophilized powder is stabilized in a lyophilized powder at a temperature of from about 5 ° C to about 25 ° C for from about 6 months to more than about 2 years, preferably greater than about 3 years, and can be easily sterilized water for injection ( Or other suitable carrier) Reconstitution is performed to provide a bendamustine liquid formulation suitable for internal administration, for example by parenteral injection. For intravenous administration, the reconstituted liquid formulation (i.e., pharmaceutical composition) is preferably a solution.

Typically, the pre-lyophilized solution or dispersion is first formulated in a pharmaceutically acceptable container by: 1) admixture with a vehicle such as mannitol (about 0 to about 50) at ambient temperature. Mg/mL) is added to water (about 65% of the total volume), 2) is mixed with about 20-35 ° C, and an organic solvent (0.5-99.9% v/v) such as TBA is added to the aqueous solution. 4) Bentonustine HCl is added to the desired concentration with mixing, 5) water is added to volume, and 6) the solution is cooled to between about 1 ° C and about 30 ° C (preferably about 5 ° C). Although the foregoing steps are presented in a particular order, those skilled in the art will appreciate that the order and magnitude of the steps can be varied if desired. The magnitude can also be based on weight.

The pre-lyophilized solution or dispersion can be sterilized prior to lyophilization, which is typically accomplished by filtration through aseptic processing, for example, through a 0.22 micron or smaller filter. Sterilization of the solution or dispersion can be achieved by other methods known in the art, such as irradiation.

In this case, the solution or dispersion was freeze-dried immediately after sterilization. Typically, the filtered solution is introduced into a sterile receiving container and then transferred to any suitable container in which the formulation can be effectively lyophilized. The formulation is typically placed in a container into which the product is intended to be sold, such as, but not limited to, in a vial as described herein and as known in the art, lyophilized.

A representative procedure for freeze-drying a pre-lyophilized solution or dispersion Listed below. However, those skilled in the art will appreciate that the procedure or method can be modified by, for example, but not limited to, pre-lyophilized solutions or dispersions and freeze-drying devices.

Initially, the product is placed in a freeze-drying chamber at a set temperature and then lowered to a temperature much lower than the freezing point of the product, typically for several hours. Preferably, the temperature is at or below about -40 ° C for at least 2 hours. After the freezing is completed, the freezing chamber and the condenser are evacuated through a vacuum pump, and the surface of the condenser tube has been previously cooled by circulating refrigerant. Preferably, the condenser tube has been previously cooled below the freezing point of the solution, preferably to about -40 ° C, more preferably to about -50 ° C or lower, even more preferably to about -60 ° C or more. low. In addition, the freezer compartment should be continuously evacuated until a pressure of from about 10 to about 600 microns, preferably from about 50 to about 150 microns, is obtained.

The product composition is then heated under vacuum in the freezer and condenser tubes. This is typically accomplished by warming the shelf in which the product is placed in the freeze-drying process in a freeze dryer process at a pressure of from about 10 to about 600 microns. The warming process should be carried out very gradually and very gradually, over a period of hours. For example, the product temperature should initially be increased from about -30 ° C to about -10 ° C and maintained for about 10-70 hours. Additionally, the product temperature can be increased from the solidification temperature for from 30 to 192 hours to from about 25 to about 40 °C. To prevent the powder of the lyophilized product from being ejected from the vial, the organic solvent and water should be completely removed during the initial drying phase. Complete drying can be determined by vacuum stabilization, condenser temperature, and product shelf temperature. After initial drying, the product temperature should be increased to between about 25 ° C and 40 ° C and maintained for about 5-40 hours.

Once the drying cycle is completed, the pressure in the freezer compartment can be sterilized, Dry nitrogen (or equivalent) is slowly released to atmospheric pressure (or slightly lower). If the product composition has been lyophilized in a container (eg, a vial), the vial can be stoppered, removed, and sealed. Several representative samples can be taken for analysis of the quality of the product for various physical, chemical and microbiological tests.

The lyophilized bendamustine formulation is typically sold as a pharmaceutical dosage form. The pharmaceutical dosage form of the invention (although in the form of a vial) can be any suitable container capable of maintaining a sterile environment, such as ampoules, syringes, co-pill bottles. Such containers may be made of glass or plastic as long as their materials do not interact with the bendamustine formulation. The seal is typically a plug, most typically a rubber stopper, preferably a bromobutyl rubber stopper, which supplies a hermetic closure.

After lyophilization, the bendamustine lyophilized powder can be filled into a container (eg, a vial), or alternatively, the pre-lyophilized solution can be filled into the vial and lyophilized therein. Forming a vial containing directly the lyophilized bendamustine formulation. After filling or lyophilizing the solution, the vials are sealed with a stopper to provide a sealed, sterilized pharmaceutical dosage form. Typically, a vial will contain about 10-500 mg per vial (preferably about 100 mg per vial) of bendamustine and about 5 mg to 2 g per vial (preferably about 170 mg/vial) lyophilized powder of mannitol.

The lyophilized formulation of the present invention may be reconstituted with water (preferably sterile water for injection) or other sterile liquid such as a cosolvent, provided to be suitable for further dilution in a suitable intravenous mixed solution, such as normal saline, for example via A solution of bendamustine administered parenterally.

B. Solubility

By visual inspection of bendamustine HCl (bendamustine) dissolved in water (alone) and with various amounts of alcohols used for lyophilization, such as methanol, ethanol, propanol, isopropanol, butyl Solubility in alcohols and third butanol (TBA). A composition of bendamustine in an amount of 15 mg/mL and mannitol in an amount of 25.5 mg/mL, dissolved in 10 mL of the specified alcohol solution, was prepared at room temperature (see Table 1). The samples were then refrigerated at 5 ° C and examined for the presence of particles and/or precipitates after 0, 3, 6 and 24 hours.

The results listed in Table 1 show that the solubility of bendamustine is dependent on the temperature and the amount of alcohol in the aqueous solution. Regarding the alcohols tested, the solubility of bendamustine increased as the concentration of alcohol increased. The formation of precipitates also depends on temperature and time. With any of the alcohols, bendamustine did not precipitate immediately, but crystallized after storage at 5 °C. The effects of each alcohol on solubility vary. Without intending to be bound by any particular theory, smaller molecular alcohols such as methanol and ethanol have less effect on solubility when compared to larger molecular alcohols (third-butanol and n-butanol). However, the shape of the alcohol is also important. For example, it has been found that n-propanol is superior to isopropanol in preventing precipitation in this system. The two alcohols having the greatest influence on solubility are n-propanol and tert-butanol.

CCS indicates clarification of colorless solution

For the quantitative determination of bendamus in three different solutions at various temperatures The experiment of the solubility of statins is summarized in Figure 1 and Table 2. The amount of TBA used in this experiment (20% (v/v) and 30% (v/v)) was based on solubility studies (results are described below). For the two solutions tested, the solubility of bendamustine was linearly decreasing with temperature from 25 °C to 0 °C. This experiment determines the data presented in Table 1 and highlights the difference in solubility of bendamustine for 20% and 30% TBA solutions.

C. Solubility

Since bendamustine is hydrolyzed and unstable in aqueous solution, it needs to be freeze-dried to produce a product suitable for medical use. However, during the manufacture of lyophilized pharmaceutical products, it is usually necessary to fill the aqueous solution prior to lyophilization. Therefore, the use of aqueous solutions during the mixing and filling of bendamustine and other nitrogen mustards can lead to degradation of the drug product. Therefore, assess the effects of various alcohols on the degradation of bendamustine to determine if a longer loading-to-finish time can be found, providing a lyophilized powder that can be reconstituted faster than existing Ribomustin® formulations, and / Or a formulation of bendamustine lyophilized formulation having a better distribution of impurities relative to specific impurities (eg, HP1 and BM1 duplexes) than Ribomustin®.

Preferably, the lyophilized preparation of the present invention is stable with respect to HP1, that is, the amount of HP1 is not slightly increased when stored at about 2 ° C to about 30 ° C (preferably 5 ° C) (not exceeding the storage) The release-half-life specification is up to 6 months, more preferably 12 months, and even more preferably more than 24 months, such as 36 months.

Table 3 lists the stability results of bendamustine in water with or without added alcohol at 24 ° C for 24 hours. Bendamustine degrades rapidly in water alone and significantly forms the hydrolysate HP1 (monohydroxybendamustine).

Another major degradation observed during this study and other long-term stability studies was the dimer of bendamustine.

Other degradation products obtained in the lyophilized product of Ribomustin are bendamustine ethyl ester (BMIEE) (formula IV) and BMIDCE (formula V). BMIEE is formed when bendamustine is reacted with ethanol.

Figure 2 summarizes the purity results of HPLC analysis of bendamustine at 5 °C after incubation in various alcohols for 24 hours. The results are expressed as a percentage of the area of the total wave front area. The values corresponding to Figure 2 are listed in Tables 3-9. The highest purity in solutions containing higher concentrations of alcohol, regardless of the alcohol what. Among the alcohols evaluated, there was minimal degradation of bendamustine in a solution containing about 30% (v/v) TBA. N-butanol is effective in preventing degradation of bendamustine in about 10% and about 20% alcohol solution. Below 20% and 30% (v/v), since n-butanol is insoluble in water at these concentrations, n-butanol in water causes a two-phase system.

Figures 3 and 4 show the amount of bendamustine degradation measured by HPLC (as described herein) by quantifying HP1 and dimer formation. HP1 and dimer formation increase with decreasing alcohol concentration, regardless of the alcohol. This increase in impurities occurs in the expected time dependence (see Table 3-9). The third-butanol and n-butanol showed superiority to other alcohols in preventing product degradation. As shown in Table 10, mannitol did not have any effect on the stability of bendamustine with TBA.

The a-di solution has 2 layers/phase liquid in the vial. The solution was vortexed prior to sample preparation.

The results in Tables 1-9 show that the stability of bendamustine HCl with HP1 and the dimers is improved as the concentration of alcohol increases.

D. Freeze drying cycle process

Different pre-lyophilized formulations were prepared at various concentrations of bendamustine, mannitol and alcohol dissolved in water. The process was changed at each step and the freezing (fast versus slow), first drying (temperature and pressure) and second drying were optimized as described herein.

Based on all of the above detailed information on solubility, stability and lyophilization, preferred formulations include the following: Water, the right amount to the desired volume

Wherein the alcohol is selected from the group consisting of methanol, n-propanol or isopropanol.

Wherein the alcohol is selected from the group consisting of methanol, n-propanol or isopropanol.

Example

The following examples are presented to illustrate specific aspects of the invention and to assist those skilled in the art to practice the invention. The examples are in no way intended to limit the scope of the invention in any way.

Material :

Bendamustine HCl, (Degussa, Lot No. 0206005 and 0206007)

Mannitol, NF or equivalent (Mallinckrodt)

Dehydrated ethanol (200 validation), USP or equivalent (Spectrum)

Third-butanol, ACS (EM Science)

Methanol (Spectrum and EMD)

Propanol (Spectrum)

Isopropyl alcohol (Spectrum)

Butanol (Spectrum)

Water, HPLC grade or equivalent (EMD)

Acetonitrile, HPLC grade or equivalent (EMD)

Trifluoroacetic acid, J.T.Baker

Methanol, HPLC grade or equivalent (EM Science, Cat# MX0488P-1)

Trifluoroacetic acid, HPLC grade or equivalent (JT Baker, Cat# JT9470-01)

Device :

Waters 2695 Alliance HPLC system with photodiode ray detector

Waters 2795 Alliance HPLC System with Dual Wavelength Detector

Analytical balances (Mettler AG285, ID#1028) and (Mettler XS205)

VirTis Freeze Dryer AdVantage

Agilent Zorbax SB-C18 5μm 80Å 4.6×250 mm column, Cat# 880975-902

Example 1 - HPLC procedure method 1

Mobile phase A: 0.1% TFA; H ₂ O

Mobile phase B: 0.1% TFA; 50% ACN: 50% H ₂ O

UV: 230 nm

Flow rate: 1.0 mL/min

Column temperature: 30 ° C

Column: Zorbax SB-C18 5μm 80Å 4.6×250 mm

Sample temperature: 5 ° C

Sample concentration: 0.25 mg/mL dissolved in MeOH

Gradient: 20% B for 1 minute 20-90% B in 23 minutes 90% B for 6 minutes Back to 20% B for 2 minutes to maintain 20% B for 4 minutes

Time: 30 minutes

After the time: 5 minutes

Method 2

Mobile phase A: 0.1% TFA; H ₂ O: CAN (9:1)

Mobile phase B: 0.1% TFA; H ₂ O: CAN (5:5)

UV: 230 nm

Flow rate: 1.0 mL/min

Column: Zorbax SB-C18 5μm 80Å 4.6×250 mm

Column temperature: 30 ° C

Sample temperature: 5 ° C

Injection volume: 10 μL

Sample concentration: 0.25 mg/mL dissolved in MeOH

Gradient: 0% B for 3 minutes 0-50% B in 13 minutes 50-70% B in 17 minutes 70-90% B in 2 minutes 90% B up to 5 minutes back to 0% B in 1 minute Maintain at 0% B for 4 minutes

Time: 40 minutes

After the time: 5 minutes

Method 3

Mobile phase A: HPLC grade water with 0.1% TFA (v/v)

Mobile phase B: HPLC grade CAN/water (1:1 v/v) with 0.1% TFA

UV: 254 nm

Flow rate: 1.0 mL/min

Column: Zorbax SB-C18 5μm 80Å 4.6×250 mm

Column temperature: 30 ° C

Sample temperature: 5 ° C

Injection volume: 5 μL

Take time: 30 minutes

After the time: 9 minutes

Thinner: methanol

gradient:

Sample Preparation - The drug product was dissolved in 200 mL MeOH. The ultrasonic wave oscillated for 6 minutes. This solution was directly injected into HPLC (ca. 0.5 mg/mL).

Method 4

Mobile phase A: HPLC grade water with 0.1% TFA (v/v)

Mobile phase B: HPLC grade CAN with 0.1% TFA (v/v)

UV: 254 nm

Flow rate: 1.0 mL/min

Column: Zorbax Bonus RP-C14 5μm 4.6×150 mm

Column temperature: 30 ° C

Sample temperature: 5 ° C

Injection volume: 2 μL

Take time: 31 minutes

After the time: 5 minutes

Thinner: NMP/0.1% TFA in water (50:50 v/v)

gradient:

Sample Preparation for Method 4 - The drug product was dissolved in a known amount of diluent to achieve a concentration of 4.2 mg/mL for direct injection into HPLC. A second dilution (100 mg/vial dose) may be required to obtain a 4.2 mg/mL sample concentration.

result

The residence time for some of the bendamustine impurities using the above HPLC method 1 is shown in Table 11. HPLC chromatograms for Ribomustin® using the HPLC procedure described herein are shown in Figure 6.

Although HPLC method 1 can resolve the impurities found in bendamustine Quality, but it cannot be separated from the possible impurities formed during the analysis, namely the methyl ester of bendamustine (BM1ME). The difference in residence time between the BM1ME and the BM1 duplex is only 0.3 minutes. To resolve the BM1 duplex, 遂 developed another HPLC method (#2). HPLC Method #2 was able to separate all impurities but required a longer duration of 45 minutes (Table 12).

The impurity profiles of various batches of bendamustine using HPLC Method 3 are listed in Table 13.

Example 2 - Solubility

Determination of bendamustine HCl (bendamustine) in water (alone) and with varying amounts of methanol, ethanol, propanol, isopropanol, butanol and tert-butanol (TBA) by visual inspection Solubility. Bendamyl statin at a concentration of 15 mg/mL and mannitol at a concentration of 25.5 mg/mL were prepared in 10 ml of the specified alcohol solution at room temperature (Table 1). The samples were then refrigerated at 5 ° C and visually observed for the presence or absence of particles and/or precipitates after 0, 3, 6 and 24 hours.

The results summarized in Table 1 show that the solubility of bendamustine is dependent on the temperature and the amount of alcohol present in the aqueous solution. For alcohols, the solubility of bendamustine increases as the concentration of alcohol increases. The formation of precipitates also depends on temperature and time.

Determination of bendamustine solubility in a 20% (v/v) TBA aqueous solution containing 25.5 mg/mL mannitol and a 30% (v/v) TBA aqueous solution containing 25.5 mg/mL mannitol (Figure) 1). Bendamustine was added to 4 mL of each solution with mixing until it no longer dissolved. The saturated solution was mixed at -8 ° C, 0 ° C, 5 ° C or 25 ° C for 1 hour. Centrifuge the sample and return it to its original temperature for at least 30 minutes. The -8 ° C sample was placed in an ice bath containing sodium chloride (to lower the temperature of the ice bath) and the temperature was measured as the sample was taken for analysis. An equal amount of each sample was taken and prepared for HPLC analysis.

The results of these experiments are shown in Figures 1 and 2. The amount of TBA used in this experiment (20% (v/v) and 30% (v/v)) (Figure 1) is based on the stability studies described herein.

As shown in Figure 1, bendamustine solubility with temperature (25 ° C to 0 °C) and linearly decreasing. The solubility of bendamustine is temperature dependent, whether or not it is dissolved in water alone or with alcohol. Due to the stability and solubility of bendamustine, 20% (v/v) TBA appears to be the lower limit required for the manufacture of potent drugs. The loading solution containing 15 mg/mL bendamustine was close to the saturation limit of 17.2 mg/mL bendamustine at 5 °C, but above the limit of 0 °C. 30% (v/v) TBA is the recommended TBA concentration for the final formulation and is well within the solubility limit, independent of temperature.

Example 3 - Stability A. Stability in water

Prepare a solution of bendamustine (15 mg/mL) and mannitol (25.5 mg/mL) in water at room temperature and immediately place in an ice bath (to quickly reduce the temperature to about 5) °C) for 10 minutes, then refrigerated at 5 °C. After storage for 0, 3, 6 and 24 hours at 5 °C, each formulation sample was analyzed by HPLC using the methods described herein.

B. Stability in alcohols

Prepared at room temperature containing 15 mg/mL bendamustine, 25.5 mg/mL mannitol and 1.9%, 5%, 10%, 20% or 30% (v/v) ethanol dissolved in water, or 5%, 10%, 20% or 30% (v/v) TBA, methanol, propanol, isopropanol or butanol in water, immediately placed in an ice bath for 10 minutes, then refrigerated at 5 ° C . After storage for 0, 3, 6 and 24 hours at 5 °C, each formulation sample was analyzed by HPLC using the methods described herein.

C. Stability results

Table 3 shows the stability results of bendamustine dissolved in water without added alcohol at 24 ° C for 24 hours. Bendamustine degrades rapidly in water, However, the stability of bendamustine increased with increasing alcohol concentration (Figures 2, 3 and 4). Although alcohols are often used in freeze drying to help solve solubility problems, the effect of alcohols on the stability of bendamustine is unique, unpredictable, and used to make bendamustine with less impurities because aqueous solutions can be used. The stability of bendamustine is still maintained. TBA was found to be the best stabilizer in the six tested alcohols (Figures 2, 3 and 4). Stored at 5 ° C for up to 24 hours, all alcohols at 30% (v / v) can reduce the formation of impurities HP1 and duplex. With regard to TBA, storage was carried out at 5 ° C for up to 24 hours, and HP 1 only reached about 0.4%. Lower concentrations of alcohol may not be effective because when loaded with 15 mg/mL bendamustine and stored at 5 ° C, there is a precipitation of bendamustine and impurities.

Example 4 - Formulation Optimization

After the solubility and stability of bendamustine have been determined, the formulation for lyophilization is optimized. Because the concentration of bendamustine in 30% TBA/water-saturated solution is higher than other alcohol solutions, the size of the vial required to fill 100 mg of bendamustine is expected to be comparable to the current Ribomustin® product. Slightly smaller. Although the saturated solution of bendamustine contained 18 mg/mL at 0 °C, the concentration of 15 mg/mL was selected to compensate for the difference in overall API purity due to the difference in batch, but a slight difference in API solubility. difference. A concentration of 15 mg/mL bendamustine was required to be 6.67 mL to fill each vial with 100 mg of bendamustine HCl.

The ratio of surface (sublimation) to volume is important for the manufacture of lyophilized products with a good appearance of rapid freeze drying. Typically, the lyophilized product will comprise from 30% to 50% by volume of the vial. A 20 mL vial with 6.67 mL contains approximately 30% of its capacity and has a surface area ratio of 0.796 cm ² /mL.

The choice of mannitol as a filler also keeps the formulation similar to Ribomustin®. A study was conducted to evaluate the effect of mannitol on the solubility of bendamustine and the appearance of the product. Mannitol reduced the solubility of bendamustine (concentration 15 mg/mL) in both ethanol and TBA aqueous solutions. For example, a solution containing 5% and 10% ethanol and TBA without mannitol does not precipitate after 24 hours. However, for samples containing mannitol (Table 1), precipitation was observed within 24 hours. In the aqueous solution containing 30% (v/v) TBA, 15 mg/mL bendamustine and 25.5 mg/mL mannitol, no precipitation occurred. In order to prevent the already formed cake from breaking during the operation, 134 mg/medical bottle of mannitol was required, and no difference was observed in the vial of up to 200 mg/vial of mannitol.

All tested alcohols increase the stability and solubility of bendamustine. However, a meaningful molar fraction is required to affect the stability and ease of manufacture of the filling solution. Smaller alcohols have an undesirable effect of lowering the freezing point of the overall solution, and thus require a long freeze-drying cycle at low temperatures. Higher concentrations of methanol and ethanol produce unpleasant and unattractive patties. Prepared 10% ethanol, 20% ethanol, 10% isopropanol, 20% isopropanol, or 30% (v) containing bendamustine (15 mg/mL), mannitol (25.5 mg/mL) /v) Aqueous TBA solution and lyophilized. A lyophilized vial filled with 10% ethanol, 20% ethanol, 10% isopropanol, 20% isopropanol solution produces a collapsed pill or film residue. The only solvent system that produces an acceptable tablet is 30% TBA. In addition, 10% ethanol, 20% ethanol, 10% isopropanol, The recovery of the lyophilized vial of 20% isopropanol was difficult and did not completely dissolve until >45 minutes.

The ability to use smaller vials is limited by the concentration or solubility of bendamustine in an aqueous/organic solution. At lower concentrations of ethanol, methanol, isopropanol, and n-propanol, which give an acceptable tablet appearance, a relatively thin solution of bendamustine is required due to solubility limitations. To maintain 100 mg of bendamustine per vial, a vial larger than 50 mL will be required. Again, the stability studies described herein show that at lower concentrations, chemical stability is not sufficient to allow for acceptable loading times.

One of the factors that influence the ease of recovery is the porosity of the lyophilizate. Generally, solid precipitated solids having a small surface area are more difficult to dissolve. Most lyophilizates containing mannitol will recover within 3-5 minutes as long as they do not form a precipitate during lyophilization, often due to evaporation of the liquid (melt back). Based on our experiments with several freeze-drying solvent systems, and without intending to be bound by any particular theory, the issue of Ribomustin® recovery may be related to precipitation caused by meltback during lyophilization. Most organic solvents are not effectively lyophilized and will melt back due to their low melting point. TBA (third butanol) has a high melting point and a similar vapor pressure compared to water. The TBA is removed by sublimation (rather than evaporation) at approximately the same rate as water. The lyophilizate prepared according to the present invention at 30% (v/v) TBA can be reconstituted in 3-10 minutes, which may take 30-45 minutes compared to commercially available Ribomustin®.

Based on solubility, stability, ease of recovery, and manufacturing considerations, the following preferred pre-lyophilized formulation of the present invention: bendamustine HCl is about 15 Mg/mL, mannitol approximately 25.5 mg/mL, approximately 30% (v/v) tert-butanol, and volume adjusted using water for injection. The formulation was then filled in amber 20 mL, 20 mm vials at 6.degree. C. and sealed with a bromobutyl stopper and placed in a pre-cooled freeze dryer.

Example 5 - Impurity Analysis

The main impurities introduced during the Ribomustin® manufacturing, mixing, filling and freeze-drying procedures, when determined by HPLC analysis (Figure 6), are the hydrolyzate of the bendamustine HP1, dimer and ethyl ester (BM1EE) . BM1EE may be formed during the manufacture of the drug, for example in a recrystallization and/or purification process. BM1EE is known to be a more potent cytotoxic drug than bendamustine. An experimental determination of whether the 30% (v/v) TBA loading of the aqueous solution resulted in the formation of bendamustine third-butyl ester.

The experiment was carried out using conventional Fisher esterification conditions for the formation of bendamustine third-butyl ester. Bendamustine was heated with HCl in 60 ° C TBA for 20 hours. No reaction was observed to occur. This result indicates that it is difficult to form the third-butyl ester of bendamustine during the refilling/finishing process. As of the current stability study, no new impurities were observed in the pharmaceutical products produced from TBA.

To help test pharmaceutical products, 遂 developed a synthetic route using a more reactive third-butyl moiety. Another intent to make a third-butyl ester is accomplished by the formation of the fluorenyl chloride of bendamustine. A suspension of bendamustine in dichloromethane was treated with chloroform chloride and N,N-dimethylguanamine. After the sulfhydryl chloride is formed, the solvent is concentrated. Add the residue to dichloromethane, tert-butanol, triethylamine and 4-dimethylaminopyridine, and mix The mixture was stirred at room temperature overnight. After all the solvent was added and purified, an unknown compound was obtained. LC-MS did not meet the molecular weight of bendamustine third-butyl ester, and proton NMR did not show a corresponding third-butyl wave front. Therefore, the use of TBA as a co-solvent has the added benefit of not forming esters from the alcohol.

Example 6 - Development of freeze-drying cycle

A number of freeze drying cycles are performed to assess the important stages of freeze drying and achieve the most efficient drying cycle. The experiment was completed to assess the effect of freezing rate, first drying temperature, time, and pressure on the product.

A. Freezing rate

The literature reports that TBA selects different crystalline forms depending on the freezing rate. In some TBA solutions, the slower the product freeze rate, the faster it dries. The larger crystals formed during slow freezing produce larger pores and enable more efficient sublimation. However, when studied with bendamustine, it was found that the freezing rate was not a decisive treatment parameter when evaluated at 2 and 8 hours.

B. First and second drying

During the first freeze-drying from the 30% TBA solution, the lyophilized cake was broken and the powder was ejected from the vial. These cakes appear to contain amorphous particles in the lyophilizate, indicating that meltback occurs. This phenomenon is reproducible and occurs when the product reaches about -10 ° C (see Figure 5), regardless of the heating rate. Test several variable numbers to determine the cause and solve the problem of powder ejection. The pressure increased from 50 μm to 150 μm during the first drying period, but powder ejection was still observed, but the degree of ejection was small. This experiment was then repeated except that the freezing rate was extended from 2 hours to 8 hours. This change has no effect.

The length of the first dryness is then evaluated. For example, evaluate the following very slow drying cycle: freezing from +25 ° C to -50 ° C in eight hours; maintaining at -50 ° C for 5 hours, heating from -50 ° C to -25 ° C in seven hours and Drying; maintaining at -25 ° C for twenty hours, heating and drying from -25 ° C to -15 ° C in two hours, and maintaining at -15 ° C for twenty hours; from -15 ° C in six hours to The temperature was raised and dried at 40 ° C and maintained at 40 ° C for twenty hours while maintaining the drying chamber pressure at 150 μm throughout the drying process. No powder ejection was observed (Fig. 5). This cycle produces a non-fragmented, fairly shaped tablet that can be easily restored. Without intending to be bound by a particular theory, problems with powder spray and difficulty in recovery may be due to the drying of the lyophilizate too quickly, thus causing strong steam to flow out of the cake and melt back. With the use of less aggressive drying cycles, reproduciblely formed aesthetically stable, stable and easily reconstitutable patties. Therefore, all unbound water and third butanol are removed prior to the second drying to prevent meltback and powder ejection. The freeze-drying cycle was further optimized under these mild conditions (Figure 5). Drying at 40 ° C for up to 20 hours resulted in the absence of immediate degradation products.

Example 7 - Freeze Drying Cycle

All of the compositions and methods disclosed herein, and the scope of their claims, may be accomplished and practiced in accordance with the present invention without undue experimentation. Although the compositions and methods of the present invention have been described in terms of a preferred embodiment, it is apparent to those skilled in the art that the compositions and methods of the present invention can be described and described without departing from the spirit and scope of the invention. The steps and steps in the method are changed in order. More precisely, it will be apparent that certain solvents which are chemically and physically related to the solvents described herein may be substituted for the solvents described herein to achieve the same or similar results. All such substitutions and modifications are obvious to those skilled in the art and are intended to be included within the spirit and scope of the invention as defined by the appended claims.

All patents, patent applications, and publications referred to in this specification are to be regarded as a All patents, patent applications, and publications are hereby incorporated by reference in their entirety in the extent of the extent of the disclosure of the disclosures of

The invention as exemplified herein is suitably carried out without any of the elements not specifically disclosed herein. Thus, for example, any of the terms "comprising," "substantially encompassing," and "consisting of" may be interchangeable with the other terms. The terms and phrases used are used for the purpose of description and not limitation, and are not intended to be Various changes may be made within the scope. Therefore, it should be understood that the present invention has been particularly disclosed by the preferred embodiments and features as desired. Modifications and variations of the presently disclosed subject matter may be employed by those skilled in the art, and it is to be understood that such modifications and variations are deemed to be included in the invention as defined by the scope of the appended claims. Within the scope.

Figure 1 shows the solubility of bendamustine at various temperatures for two different solutions of bendamustine dissolved in t-butanol.

Figure 2 shows the purity results of HPLC analysis of bendamustine in various alcohols after 24 hours at 5 °C. The results are expressed as a percentage of the area of the bendamustine peak.

Figure 3 shows the formation of HP1 (Formula II) after 24 hours at 5 °C in various alcohol/water cosolvents.

Figure 4 shows the formation of the dimer (formula III) after 24 hours at 5 °C in various alcohol/water cosolvents.

Figure 5 shows a freeze-drying cycle for the use of TBA/water cosolvent for bendamustine.

Figure 6 shows the chromatogram of HPLC method No. 1 for Ribomustin®.

Claims (40)

A method of preparing a lyophilized preparation of bendamustine or bendamustine hydrochloride, comprising: a) dissolving bendamustine or bendamustine hydrochloride in a stable concentration Pre-freeze-dried solution is formed by including about 20% to 30% (v/v) of one or more alcohol solvents of methanol, ethanol, propanol, isopropanol, butanol and butanol. (pre-lyophilization solution); and b) lyophilizing the pre-lyophilized solution; wherein the bendamustine lyophilized formulation contains no more than about 0.5 area% of HP1, Where HP1 is the amount of HP1 at the time of release. The method of claim 1, wherein the alcohol is ethanol. The method of claim 2, wherein the concentration of the ethanol is about 30%. The method of claim 1, wherein the alcohol is third butanol. The method of claim 4, wherein the concentration of the third butanol is about 30%. The method of claim 1, wherein the excipient is added prior to lyophilization. The method of claim 6, wherein the excipient is mannitol. The method of claim 1, wherein the bendamustine concentration is about 2 to 50 mg/mL. The method of claim 8, wherein the alcohol is third butanol. The method of claim 9, wherein the concentration of the third butanol is about 30%. The method of claim 8, wherein the alcohol is ethanol. The method of claim 11, wherein the concentration of the ethanol is about 30%. The method of claim 1, wherein the step b) comprises: i) freezing the pre-lyophilized solution to a temperature below about -40 ° C to form a frozen solution; ii) maintaining the frozen solution At or below about -40 ° C for at least 2 hours; iii) ramping the frozen solution to a first drying temperature between about -40 ° C to about -10 ° C to form Drying the solution; iv) maintaining for about 10 to about 70 hours; v) increasing the dried solution to a second between about 25 ° C and about 40 ° C The secondary drying temperature; and vi) is maintained for about 5 to about 40 hours to form the bendamustine lyophilized formulation. The method of claim 13, wherein the alcohol is ethanol. The method of claim 14, wherein the concentration of the ethanol is about 30%. The method of claim 13, wherein the alcohol is third butanol. The method of claim 16, wherein the concentration of the third butanol is about 30%. The method of claim 1, wherein the step b) comprises: i) freezing the pre-lyophilized solution to about -50 ° C to form a frozen solution; ii) maintaining the frozen solution at about -50 At least 2 hours to about 4 hours at ° C; iii) gradually increasing to a first drying temperature between about -20 ° C to about -12 ° C to form a dry solution; iv) maintaining about 10 to about 10 at a first drying temperature 48 hours; v) increasing the dried solution to a second drying temperature of between about 25 ° C and about 40 ° C; and vi) maintaining the second drying temperature for at least 5 hours up to about 20 hours. The method of claim 18, wherein the alcohol is ethanol. The method of claim 19, wherein the concentration of the ethanol is about 30%. The method of claim 18, wherein the alcohol is third butanol. The method of claim 21, wherein the concentration of the third butanol is about 30%. The method of claim 1, wherein the step b) comprises: i) feeding at a shelf temperature of about 5 ° C; ii) freezing to about -50 ° C over a period of about 8 hours. ; iii) maintained at -50 ° C for about 4 hours; iv) gradually increased to about -20 ° C over a period of about 3 hours; v) maintained at about -20 ° C for 6 hours; vi) lasted about 1 hour During the period, the temperature is gradually increased to about -15 ° C; vii) is maintained at -15 ° C for about 20 hours; viii) gradually increased to -12 ° C over a period of about 0.5 hours; ix) maintained at about -12 ° C. 15.5 hours; x) gradually increasing to between about 25 ° C and about 40 ° C or higher during a period of about 15 hours; xi) maintaining between about 25 ° C and about 40 ° C for about 10 hours; xii) Gradually increasing to about 40 ° C over a period of about 1 hour; xiii) maintaining at about 40 ° C for about 5 hours; and xiv) at about 5 ° C, at a pressure of about 13.5 psi, unloading in medicine In the accepted container, it is sealed; wherein the pressure throughout the first drying step iv, v, vi, vii, viii and ix is about 150 microns, and the second drying step x, xi, xii and xiii is about 50 Micron. According to the method of claim 23, wherein step (xi) is gradually warmed to about 30-35 ° C for about 15 hours. According to the method of claim 24, wherein the alcohol is Third butanol. The method of claim 25, wherein the concentration of the third butanol is about 30%. The method of claim 24, wherein the alcohol is ethanol. The method of claim 27, wherein the concentration of the ethanol is about 30%. A formulation for lyophilization comprising bendamustine at a concentration of about 15 mg/mL, mannitol at a concentration of about 25.5 mg/mL, and a third concentration at a concentration of about 30% (v/v) Alcohol and water. A formulation for lyophilization comprising bendamustine at a concentration of about 15 mg/mL, mannitol at a concentration of about 25.5 mg/mL, and ethanol and water at a concentration of about 30% (v/v) . Use of a lyophilized preparation prepared according to the method of any one of claims 1 to 28 for the preparation of a medicament for treating a medical condition of a patient. A use according to the formulation of claim 29 or 30 for the preparation of a medicament for treating a medical condition in a patient. According to the use of claim 31 or 32, wherein the condition is selected from the group consisting of chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, breast cancer, small cell lung cancer and Autoimmune disease. According to the use of claim 33, wherein the condition is non-Hodgkin's lymphoma. According to the use of the scope of claim 33, wherein the condition is chronic lymphocytic leukemia. According to the use of the scope of claim 33, wherein the condition is multiple myeloma. The use according to claim 31 or 32, wherein the medicament further comprises one or more antitumor agents. The use according to claim 37, wherein the anti-tumor agent is an antibody specific for CD20. The use according to claim 33, wherein the autoimmune disease is rheumatoid arthritis, multiple sclerosis or lupus. The use according to claim 31 or 32, wherein the medical condition is a hyperproliferative disorder.